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The Mechanical Unfolding of Fibronectin Using Atomic Force Microscopy and Its Relevance to Biocompatibility Studies

Meadows, Pamela Yvonne
(2005)
The Mechanical Unfolding of Fibronectin Using Atomic Force Microscopy and Its Relevance to Biocompatibility Studies.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)

Abstract

Protein adsorption to surfaces has hindered advancements made in biocompatible implants and drug delivery. Once a protein adsorbs, it can undergo conformational changes and unfold to expose buried cryptic sites of the protein. These sites are often critical for signaling additional proteins and cells to adsorb to the surface which can eventually lead to an implant's failure or the destruction of drug delivering devices. To improve the biocompatibility of these implants, an understanding of how a surface affects a protein's conformation and stability is required. In this work, an atomic force microscope (AFM) is used to quantify the stability of fibronectin (FN), an adhesion promoting protein, on mica, gold, poly(ethylene glycol), and -CH3, -OH, and -COOH terminated alkanethiol self-assembled monolayers. The thermodynamic parameters associated with this mechanically induced denaturation are presented as a function of surface type and amount of adsorbed protein using two different models. Results indicate greater stabilization of FN in densely deposited films while greater surface denaturation occurs as the proteins become more isolated on the substrate. Additional information about the protein's binding state was also obtained. Proteins aggregated on a hydrophobic surface adopted more rigid conformations apparently as a result of increased surface denaturation and tighter binding while looser conformations were observed on more hydrophilic surfaces. Finally, the force spectroscopy experiments were examined for any biocompatibility correlation by seeding substrates with human umbilical vascular endothelial cells (HUVEC). As predicted from the models used in this work, surfaces with aggregated FN promoted cellular deposition while surfaces with proteins sparsely populated hindered cellular deposition and growth. The AFM's use as a means for projecting cell deposition and perhaps biocompatibility does look promising.